1. Field of the Invention
The present invention relates to a method for the detection and identification of macromolecules of interest associated with particles. More particularly the invention relies upon the diffusion of macromolecules from the particle, and their binding in proximity to the particle to enable the macromolecules to be identified as belonging to a particular class of particles. The method may also include the identification of the particles from which the macromolecules are diffused.
2. Description of Related Art
There are many situations in which particles in a fluid stream may cause disease, environmental damage or be of interest for some other reason. In some case, the fluid stream may be carrying a large number of different particles. In this case it may be difficult to determine which of the particles are causing the effect being studied. A particular example is that asthma is believed to be associated with airborne allergens. There may, however, be many airborne allergens in the air but only one or a few may be causing the asthma.
Asthma is a chronic disease affecting all age groups in the community. The study of asthma has been hindered by the shortage of suitable methods to identify and measure the characteristics of allergen exposure which is relevant to the disease. These characteristics include:
(1) the identify of the allergen; PA1 (2) the quantity of the allergens; and PA1 (3) the size of the particles which are the allergen source. PA1 a) collecting the particles onto a surface; PA1 b) causing the macromolecular species, if present, to diffuse out of the particles; PA1 c) immobilising non-specifically the diffused macromolecules in close proximity to the particles; and PA1 d) detecting the presence or absence of the macromolecules while maintaining the close proximity of the bound macromolecules, if any, and the particles from which they diffused. PA1 a) collecting the particle onto a surface; PA1 b) causing soluble macromolecules associated with the particles to diffuse out of the particles; PA1 c) immobilising non-specifically at least some of the possibly antigenic or allergenic macromolecules that diffused out of the particles in close proximity to the particles; PA1 d) detecting those immobilised macromolecules that are antigenic or allergenic to the patient; and PA1 e) identifying the particle associated with the macromolecules and from which the macromolecules diffused.
It may be desired to know which of the particles present in the patient's environment are responsible for the observed symptoms. This may allow a determination of the source of the allergens and suggest mechanisms for prophylactically treating the condition.
It has long been conventional to estimate exposure to pollen allergens by collecting pollen particles on sticky surfaces, identifying them by morphological criteria and individually counting them. Mould exposure has been similarly monitored by biological and/or morphological methods. In each of these cases the identifiable biological particles are used as a proxy for the quality of allergens present in the sampled environment. These methods require highly skilled operators and are very labour intensive. They also suffer from the problem that the presence of a particular particle does not indicate that that particle is the source of the allergen of interest.
Immunoassays of allergens from many sources (pollens, mites, fungi, animal dander, etc.) based on aqueous extraction of material collected on filters and traps has the advantages of speed and simplicity if suitable specific antisera is available. These methods also allow quantification of the exposure to the specific allergens. There is, however, no one-to-one relationship established by such methods between specific allergens and individual particles, as the allergens are extracted from the entire sample of collected particles. There is thus no ability to associate a specific type of particle with sensitisation of the patient in a way commonly associated with an observed disease state.
The known methods of particle detection and analysis do not readily allow the provision of an answer to the most fundamental question about characterising allergen exposure i.e., "What aeroallergens are present that an individual is allergic to?". Skin-prick testing, RAST assays and questioning by a physician cannot definitely elucidate what allergens are producing the observed symptoms. In some cases, using such tests may be quite misleading as they cannot determine the time when an individual was exposed to the sensitising allergen nor can they detect sensitisation to allergens not in the test panel.
Not only is it important in a number of clinical situations to identify a specific offending allergen, it is also impossible in a more general sense to be able to make a cross sectional study of an exposure situation to understand the composition of different allergens which may be present. There are no methods which specifically focus on identifying allergen sources per se. This takes on added importance in an era when allergic disease is said to be increasing, yet we are unable to probe the environment to know if there are new, as yet unidentified, allergens present.
It has been shown in Tovey ER et al, Am. Rev. Resp. Dis. 1981; 124:630-635 that individual airborne mite faecal particles can be identified by a ring of immunoprecipitation developed around them following their impaction onto a bed of agarose containing allergen specific antibody. The identification of the particles was made by microscopy. Problems associated with this system are that only very short exposure times of the agarose bed is possible and that only specific polyclonal high-titre (hyper immune) antisera specific for a predetermined antigen can be used. Such high-titre sera is normally only available from an animal, such as a rabbit, that has been repeatedly injected with the allergen or antigen of interest and an adjuvant. The method would not work directly with IgE antibodies in the serum of an allergic person or with monoclonal antibodies. In neither of these cases is a visible immunoprecipitate formed.
The feasibility of using nitrocellulose membranes to press-blot antigens from individual particles has been described in Schumacker et al. J. Al. Clin.Imm. 1988; 82:608-16. In this method the particles collected and the nitrocellulose allergen blot are separated so that the immunostained antigens cannot be associated with the specific particles from which they originated.